The vocal fold extracellular matrix (VF-ECM) consists of a complex three-dimensional protein scaffold that is biomechanically favorable for voice production. Disruption of this protein matrix has severe consequences for vocal fold oscillation and generally results in a dysphonia that is difficult to treat effectively with current medical and surgical interventions. Lack of progress in the treatment of VF-ECM based disorders is in part due to limited understanding of the native biological structure and function of the matrix, and the manner in which it is altered under given physiological and disease states. Previous work has addressed the role of individual and small groups of proteins in VF-ECM structure and function;however there remains a need for an overarching and unifying understanding of how these individual players interact to form a functional biological and biomechanical system. A proteomic approach to VF-ECM research offers the means to simultaneously profile every protein within the matrix, and in doing so, represent the entire functional output of the system. Our long-term goal is to construct a fully annotated proteomic map of the VF-ECM, elucidate the proteomic alterations that accompany specific VF-ECM disease states, and construct a proteomic blueprint for the engineering of acellular and synthetic VF-ECM scaffolds for therapeutic purposes. Our overarching long- term hypothesis is that disease specific alterations in the VF-ECM are the result of proteome-wide changes involving the cell-mediated coordination of functionally interdependent proteins. Our short-term hypothesis, which we will test in the research outlined in this proposal, is that the native (cell populated) VF-ECM and decellularized VF-ECM have distinct and characteristic proteomic profiles. This work will be completed using a rat model and will utilize two complementary proteomic methodologies: (a) Protein separation by two- dimensional sodium dodecyl sulfate polyacrylamide gel electrophoresis (2D SDS-PAGE), and (b) protein species identification by mass spectrometry (MS) fingerprinting, utilizing matrix assisted laser desorption/ionization - time of flight mass spectrometry (MALDI-TOF MS). Our specific aims are: (Aim 1) To establish an annotated 2D SDS-PAGE reference map of the rat VF-ECM proteome;and (Aim 2) to characterize and compare the proteomic profiles of native (cell populated) VF-ECM and decellularized VF- ECM. Completion of these aims will result in a draft proteomic reference map for ongoing VF-ECM research, and a proteomic blueprint for VF-ECM tissue engineering strategies based on the repopulation and manipulation of decellularized VF-ECM scaffolds. This work is highly significant as it will facilitate the investigation of system-wide changes in protein expression following any form of VF-ECM manipulation, physiological perturbation or disease process;and will directly inform future vocal fold tissue engineering efforts. We anticipate that the mapping data generated in this project will be highly valuable to other scientists and have included a data sharing plan to make our raw data available to the scientific community. Project Narrative: This project will result in the development of a research tool that promises to improve our understanding of the network of proteins inside the vocal folds that facilitate vibration for voice production. Using this tool, we will be able to improve our understanding of how the entire network of vocal fold proteins work in concert and how this network is altered by vocal fold disease. This project will also result in the development of a blueprint for the engineering of a healthy protein matrix for restoring damaged vocal folds.